Compositions and methods for promoting neuronal outgrowth

ABSTRACT

Neural outgrowth in the central nervous system is achieved by administering chondroitinase AC and/or chondroitinase B to degrade chondroitin sulfate proteoglycans that inhibit or contribute to the inhibition of nervous tissue regeneration.

CROSS-REFERENCE TO RELATED APPLICATION

Priority to U.S. Provisional Application Ser. No. 60/377,669 filed May4, 2002 is hereby claimed.

BACKGROUND

1. Technical Field

This disclosure relates to methods for promoting neurite outgrowth afternerve cell loss as a result of central nervous system (“CNS”) injury ordisease. In particular, chondroitinase AC and chondroitinase B are usedto promote neurite outgrowth.

2. Description of Related Art

After a spinal cord injury in the adult mammalian central nervous system(CNS), the inability of axons to regenerate may lead to permanentparalysis. An injury-caused lesion will develop glial scarring, whichcontains extracellular matrix molecules including chondroitin sulfateproteoglycans (CSPGs). CSPGs inhibit nerve tissue growth in vitro, andnerve tissue regeneration at CSPGs rich regions in vivo.

A number of molecules, and specified regions thereof, have beenimplicated in the ability to support the sprouting of neurites from aneuronal cell, a process also referred to as neurite outgrowth. The termneurite refers to both axon and dendrite structures. This process ofspouting neurites is essential in neural development and regeneration,especially after physical injury or disease has damaged neuronal cells.Neurites elongate profusely during development both in the central andperipheral nervous systems of all animal species. This phenomenonpertains to both axons and dendrites. However, adult neurite regrowth inthe CNS is increasingly lost with evolutionary progress.

Various polypeptides, especially cell adhesion molecules (CAMs), havebeen known to promote neural cell growth. While early efforts in thisarea of research concentrated on the adhesion-promoting extracellularmatrix protein fibronectin (FN), other polypeptides have also been foundto promote neural growth. For example, U.S. Pat. No. 5,792,743,discloses novel polypeptides and methods for promoting neural growth inthe central nervous system of a mammal by administering a soluble neuralCAM, a fragment thereof, or a Fc-fusion product thereof. U.S. Pat. No.6,313,265 discloses synthetic polypeptides containing thepharmacologically active regions of CAMs that can be used in promotingnerve regeneration and repair in both peripheral nerve injuries as wellas lesions in the central nervous system.

While helpful, the use of regenerative proteins alone may not besufficient to effect repair of a damaged nervous system.

One area that has been determined to be of significance in the repairand regeneration of cellular tissue, including neural tissue, is theextracellular matrix. Extracellular matrix proteins (“EMPs”) are foundin spaces around or near cells of multicellular organisms and aretypically fibrous proteins of two functional types: mainly structural,e.g., collagen and elastin, and mainly adhesive, e.g., fibronectin andlaminin.

During approximately the past two decades, the base knowledge of celladhesion and migration in extracellular matrices (ECMs) at the molecularlevel has expanded rapidly. The action of enzymes and other polypeptideswhich degrade components of the extracellular matrix and basementmembranes may facilitate the events of neural repair by a variety ofmechanisms including the release of bound cytokines and by increasingthe permeability of the matrix, thereby enhancing the mobility ofmediator molecules, growth factors and chemotactic agents, as well asthe cells involved in the healing process. For example, U.S. Pat. No.5,997,863 discloses the use of glycosaminoglycans to manipulate cellproliferation and promote wound healing.

ECM molecules include the inhibitory CSPGs. Components of the CSPGs havebeen identified as the glycosaminoglycans, chondroitin sulfate (CS) anddermatan sulfate (DS). Removal of these inhibitory molecules would allowneurites to regenerate and reinnervate an area after physical injury ordisease.

Previous studies have found that chondroitinases can lyase and degradeCSPGs and, including, CS and DS. One study found that chondroitinase ABCremoved glycosaminoglycan (GAG) chains in and around lesioned areas ofrat CNS, in vivo. The degradation of GAGs promoted expression of agrowth-associated protein, GAP-43, indicating increased regenerativepropensity in treated cells. However, this growth-associated protein isassociated with regeneration in peripheral, but not central, nerveinjuries. Applications of chondroitinase ABC to an injured corticospinaltract (CST) prevented axon retraction from the affected area andpromoted more axon fiber growth than the control, with some axonsarborizing into gray matter. Regenerated CST axons establishedfunctional connections. (Bradbury et al., Chondroitinase ABC promotesfunctional recovery after spinal cord injury, Nature 416: 636-640(2002)). Another study found that in vitro chondroitinase ABC treatmentof rat spinal cord regenerated neurons on a tissue section substrata.This study observed that degradation of CSPGs may promote theneuro-stimulatory effects of laminin. (Zuo et al. Degradation ofchondroitin sulfate proteoglycan enhances the neurite-promotingpotential of spinal cord tissue, Exp. Neurol. 154(2): 654-62 (1998)). Ina later study by same primary researcher, it was reported that injectionof chondroitinase ABC at the site of nerve damage degraded CSPGs andincreased the ingress of axonal sprouts into the basal laminae of thedistal nerve segment, which may be by enabling more latitude in growthat the interface of coapted nerve. (Zuo et al. Regeneration of axonsafter nerve transaction repair is enhanced by degradation of chondroitinsulfate proteoglycan. Exp. Neurol. 176(1): 221-8 (2002)). The same groupof researchers also found chondroitinase ABC treatments regeneratedaxons on into acellular grafts at a much higher rate than the controlgrafts. (Krekoski et al., Axonal regeneration into acellular nervegrafts is enhanced by degradation of chondroitin sulfate proteoglycan.J. Neurosci. 15:21(16): 6206-13 (2001)).

The use of chondroitinase AC and chondroitinase B would be advantageousto promote neurite growth in mammals because these chondroitinasesstrongly promote neurite outgrowths directly in the CNS, itself, as wellas in the peripheral nervous system.

SUMMARY

Neurite outgrowth is promoted by administering chondroitinase AC,chondroitinase B or a mixture thereof to an injured area of the centralnervous system.

Various types of chondroitinase AC, and chondroitinase B can beadministered to a mammal afflicted with a CNS injury, whether the injuryis immediate or long-standing. The chondroitinase is administered inamount effective to degrade CSPGs and thereby promote neurite outgrowth.

The chondroitinases can be administered with a suitable pharmaceuticalcarrier. The administration can be topical, local or systemic.

The administration of chondroitinases AC and/or chondroitinase B and theresulting promotion of neural growth in accordance with this disclosurerestores motor and sensory functions, to varying degrees, depending onthe responsiveness of each individual.

DETAILED DESCRIPTION

The present disclosure is directed to a method of treatment formammalian central nervous system injuries, typically caused by trauma ordisease. In particular, Chondroitinase AC and chondroitinase B,individually and in combination, provide a therapeutic treatment forspinal cord injuries. The phrase “spinal cord injuries” as used hereinincludes disease and traumatic injuries, such as severing or crushing ofneurons brought about by an auto accident, fall, knife or bullet wound,as well as other injuries. Practice of the present methods will conferclinical benefits to the treated mammal, providing clinically relevantimprovements in at least one of the subject's motor coordinationfunctions and sensory perception. Clinically relevant improvements canrange from a detectable improvement to a complete restoration of animpaired or lost central nervous system.

After a spinal cord injury in the adult mammalian central nervous system(CNS), the inability of axons to regenerate may lead to permanentparalysis. The site of the CNS spinal cord injury develops a lesion orglial scar by an increase in the deposition of extracellular matrixmolecules by astrocytes and oligodendrocytes at the site of injury.These extracellular matrix molecules include chondroitin sulfateproteoglycans (CSPGs), which are highly expressed in scarring areas.CSPGs inhibit nerve tissue growth in vitro, and nerve tissueregeneration at CSPGs rich regions in vivo. Chondroitin sulfates A, Band C are the predominant forms found in mammals. These chondroitins maybe involved in modulation of various biological activities includingcell differentiation, adhesion, enzymatic pathways, and hormoneinteractions. The presence of chondroitin sulfate proteoglycans iselevated in the later stages of cell growth in response to tissue andvessel damage.

The glycosaminoglycans (GAGs), chondroitin sulfate (CS) and dermatansulfate (DS), are important components of CSPG. They are inhibitorymolecules that contribute to the lack of regeneration of the CNS inadult mammals, by hindering axonal and neuritic growth. (However, CSPGsare important in neuronal guidance and patterning during development,rather than inhibition).

Glycosaminoglycans are unbranched polysaccharides consisting ofalternating hexosamine and hexuronic residues which carry sulfate groupsin different positions. The GAGs are typically divided into threefamilies according to the composition of the disaccharide backbone.These are: heparin/heparan sulfate [HexA-GlcNAc(SO.sub.4)]; chondroitinsulfate [HexA-GalNAc]; and keratan sulfate [Gal-GlcNAc]. The chondroitinsulfate family includes seven sub-types designated unsulfatedchondroitin sulfate, oversulfated chondroitin sulfate, and chondroitinsulfates A-E, which vary in the number and position of their sulfatefunctional groups. Chondroitin sulfate B is also referred to as dermatansulfate, and it differs in that iduronic acid is the predominant residuein the alternative hexuronic acid position.

It has now been found that the chondroitin enzymes chondroitinase AC andchondroitinase B are useful in controlling and/or inhibiting the effectsof chondroitin sulfates and in developing therapeutics for the treatmentof disease states.

Chondroitinase AC and chondroitinase B are chondroitin lyase enzymes,which may be derived from various sources. Any chondroitinase AC or Bmay be used in the disclosure, including, but not limited tochondroitinase AC (derived from Flavobacterium heparinum; T. Yamagata,H. Saito, O. Habuchi, S. Suzuki, J. Biol. Chem., 243, 1523 (1968));chondroitinase AC II (derived for Arthobacter aurescens; K. Hiyama, S.Okada, J. Biol. Chem., 250, 1824 (1975), K. Hiyama, S. Okada, J.Biochem. (Tokyo), 80, 1201 (1976)); chondroitinase AC III (derived fromFlavobacterium sp. Hp102; H. Miyazono, H. Kikuchi, K. Yoshida, K.Morikawa, K. Tokuyasu, Seikagaku, 61, 1023 (1989)); chondroitinase B(derived from Flavobacterium heparinum; Y. M. Michelaaci, C. P.Dietrich, Biochem. Biophys. Res. Commun., 56, 973 (1974), Y. M.Michelaaci, C. P. Dietrich, Biochem. J., 151, 121 (1975), K. Maeyama, A.Tawada, A. Ueno, K. Yoshida, Seikagaku, 57, 1189 (1985)); andchondroitinase B (derived from Flavobacterium sp. Hp102; H. Miyazono, H.Kikuchi, K. Yoshida, K. Morikawa, K. Tokuyasu, Seikagaku, 61, 1023(1989)). Suitable chondroitinase AC and chondroitinase B arecommercially available from Seikagaku America, Falmouth, Mass., USA.Additionally, the enzymes may be produced by the methods disclosed inU.S. Pat. No. 6,093,563 by Bennett et al., the disclosure of which isincorporated herein.

Chondroitinase enzyme activity can be stabilized by the addition ofexcipients or by lyophilization. Stabilizers include carbohydrates,amino acids, fatty acids, and surfactants and are known to those skilledin the art. Examples include carbohydrate such as sucrose, lactose,mannitol, and dextran, proteins such as albumin and protamine, aminoacids such as arginine, glycine, and threonine, surfactants such asTWEEN® and PLURONIC®, salts such as calcium chloride and sodiumphosphate, and lipids such as fatty acids, phospholipids, and bilesalts. The stabilizers are generally added to the protein in a ratio of1:10 to 4:1, carbohydrate to protein, amino acids to protein, proteinstabilizer to protein, and salts to protein; 1:1000 to 1:20, surfactantto protein; and 1:20 to 4:1, lipids to protein. Other stabilizersinclude high concentrations of ammonium sulfate, sodium acetate orsodium sulfate, based on comparative studies with heparinase activity.The stabilizing agents, preferably the ammonium sulfate or other similarsalt, are added to the enzyme in a ratio of 0.1 to 4.0 mg ammoniumsulfate/IU enzyme.

Chondroitinase may be administered topically, locally or systemically.Topical or local administration is preferable for greater control ofapplication. The chondroitinases, singularly or in combination, can bemixed with an appropriate pharmaceutical carrier prior toadministration. Examples of generally used pharmaceutical carriers andadditives are conventional diluents, binders, lubricants, coloringagents, disintegrating agents, buffer agents, isotonizing agents,preservants, anesthetics and the like. Specifically pharmaceuticalcarriers that may be used are dextran, sucrose, lactose, maltose,xylose, trehalose, mannitol, xylitol, sorbitol, inositol, serum albumin,gelatin, creatinine, polyethylene glycol, non-ionic surfactants (e.g.polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hardenedcastor oil, sucrose fatty acid esters, polyoxyethylene polyoxypropyleneglycol) and similar compounds.

Pharmaceutical carriers may also be used in combination, such aspolyethylene glycol and/or sucrose, or polyoxyethylene sorbitan fattyacid esters, polyoxyethylene sorbitan monooleate (20 E. O.) isparticularly preferred.

The treatment regimen according to the invention is carried out by ameans of administering chondroitinase AC and/or chondroitinase B to thelesions of the injured area of the CNS. The mode of administration, thetiming of administration and the dosage are carried out such that thefunctional recovery from impairment of the CNS is enhanced by thepromotion of neurite outgrowth. The treatments of the present disclosuredeliver an effective amount of chondroitinase AC and/or chondroitinase Bto the injured site. The term “effective amount” means an amountsufficient to degrade the CSPGs of the lesioned area of the spinal cord.The effective amount of chondroitinase can be administered in a singledosage, two dosages or a plurality of dosages. In a preferredembodiment, the dosage is administered within 12 hours after injury, oras soon as is feasible. In another embodiment, the dosage isadministered to an injured mammal in one, two or a plurality of dosages;such dosages would be dependant on the severity of the injury and theamount of CSPGs present in the glial scarring. Where a plurality ofdosages is administered, they may be delivered on a daily, weekly, orbi-weekly basis. The delivery of the dosages may be by means of catheteror syringe. Alternatively, the treatment can be administered duringsurgery to allow direct application to the glial scar.

Once the chondroitinases are administered, the degradation of CSPGsremoves the inhibitory molecules that block neurite outgrowth, and allowthe regeneration of neurites into the affected area. The chondroitinaseAC and chondroitinase B degrade CS and DS, respectively, resulting inunsaturated sulfated disaccharides. Chondroitinase AC cleaves CS at 1, 4glycosidic linkages between N-acetylgalactosamine and glucuronic acid inthe polysaccharide backbone of CS. Cleavage occurs throughbeta-elimination in a random endolytic action pattern. Chondroitinase Bcleaves the 1, 4 galactosamine iduronic acid linkage in thepolysaccharide backbone of DS. The cleavage of both CS and DS occursthrough a beta-elimination process which differentiates these enzymaticmechanisms from mammalian GAG degrading enzymes.

The removal of CS and DS from the glial scar permits the regeneration ofneurite outgrowths into the injured area.

The regeneration of the nerve cells in to the affected CNS area allowsthe return of motor and sensory function. Clinically relevantimprovement will range from a detectable improvement to a completerestoration of an impaired or lost nervous function, varying with theindividual patients and injuries.

Although preferred and other embodiments of the invention have beendescribed herein, further embodiments may be perceived by those skilledin the art without departing from the scope of the invention as definedby the following claims.

1. A method of promoting neurite outgrowth of central nervous systemneural cells in a mammal comprising contacting injured central nervoussystem neural cells directly at the site of a central nervous systeminjury with a composition comprising a compound selected from the groupconsisting of chondroitinase AC and chondroitinase B; whereby neuriteoutgrowth occurs from said central nervous system neural cells.
 2. Amethod as in claim 1, wherein the compound is chondroitinase AC.
 3. Amethod as in claim 1, wherein the compound is chondroitinase B.
 4. Amethod as in claim 1, wherein the composition further comprises apharmaceutically acceptable carrier.
 5. A method as in claim 1, whereinthe composition further comprises a stabilizer.
 6. The method of claim1, wherein the site of the central nervous system injury is the spinalcord.
 7. The method of claim 1, wherein the mammal is a human.